Chronic Alcohol Feeding Research Articles

Alcohol-mediated miR-34a modulates hepatocyte growth and apoptosis.

MicroRNAs (miRs) have been recently shown to be heavily involved in the development of alcoholic liver disease (ALD) and suggested as a potential therapeutic target in ALD. The miR‐34a was consistently reported to be significantly elevated in several ALD rodent models, but it remains unclear how miR‐34a modulates the cellular behaviours of hepatocytes in ALD development and progression. This study aims to characterize alcohol‐induced miR‐34a impact on hepatocytes growth and apoptosis. The miRNA array was performed to assess changes in miRNA after chronic alcohol feeding. Liver and blood samples were used to examine ALD progression. The miR‐34a was overexpressed in human hepatocytes to evaluate its impact on cell growth and apoptosis. Real‐time quantitative PCR and Western blot were used to determine the growth and apoptosis molecular signalling pathways associated with miR‐34a. Alcohol feeding significantly promoted fatty liver progression, serum ALT levels, apoptosis and miR‐34a expression in rat liver. Overexpression of miR‐34a in human hepatocytes suppressed cell growth signallings, including c‐Met, cyclin D1 and cyclin‐dependent kinase 6 (CDK6). The miR‐34a might also inhibit the expression of sirtuin 1 (Sirt1) and its target, B‐cell lymphoma 2. Interestingly, the expression of miR‐34a reverses the suppressive effects of ethanol on cell growth. But, miR‐34a promotes hepatocyte senescence and apoptosis. Although the miR‐34a‐mediated down‐regulation of cell growth‐associated genes may contribute to cell growth retardation, other miR‐34a targets, such as Sirt1, may reverse this phenotype. Future studies will be needed to clarify the role of miR‐34a in ALD progression.

Liver kinase B1/AMP-activated protein kinase-mediated regulation by gentiopicroside ameliorates P2X7 receptor-dependent alcoholic hepatosteatosis.

Regulating P2X7 receptor-mediated activation of NLRP3 inflammasomes could be a therapeutic strategy to treat alcoholic hepatosteatosis. We investigated whether this process was modulated by gentiopicroside, the main active secoiridoid glycoside from Gentiana manshurica Kitagawa. In vivo models of acute and chronic alcoholic hepatosteatosis were established by intragastrically administered ethanol or using chronic plus binge ethanol feeding of Lieber-DeCarli liquid diet to male C57BL/6 mice. In vitro, HepG2 cells were treated with ethanol. RAW 264.7 macrophages and murine bone marrow-derived macrophages (BMDMs) were stimulated with LPS and ATP. In both the acute and chronic alcohol-induced mouse hepatosteatosis models, gentiopicroside decreased serum aminotransferases and triglyceride accumulation. Up-regulated SREBP1, down-regulated PPARα and phosphorylated acetyl-CoA carboxylase caused by acute and chronic alcohol feeding were modulated by gentiopicroside, through the elevation of LKB1 and AMPK. Suppression of P2X7 receptor-NLRP3 activation by gentiopicroside inhibited IL-1β production. In ethanol-exposed HepG2 cells, gentiopicroside reduced lipogenesis and promoted lipid oxidation via activation of P2X7 receptor-NLRP3 inflammasomes. Genetic or pharmacological blockade of P2X7 receptors enhanced AMPK activity and reduced SREBP1 expression in ethanol-treated HepG2 cells. Gentiopicroside down-regulated P2X7 receptor-mediated inflammatory responses in LPS/ATP-stimulated RAW 264.7 macrophages and BMDMs. IL-1β from macrophages accelerated lipid accumulation in hepatocytes. Depleting macrophages by clodronate liposomes ameliorated alcoholic hepatosteatosis, and it was further alleviated by gentiopicroside. Activation of LKB1/AMPK signalling by gentiopicroside was mediated by the P2X7 receptor-NLRP3 inflammasome, suggesting the therapeutic value of blocking P2X7 receptors in the treatment of alcoholic hepatosteatosis.

Cannabidiol attenuates alcohol-induced liver steatosis, metabolic dysregulation, inflammation and neutrophil-mediated injury

Cannabidiol (CBD) is a non-psychoactive component of marijuana, which has anti-inflammatory effects. It has also been approved by FDA for various orphan diseases for exploratory trials. Herein, we investigated the effects of CBD on liver injury induced by chronic plus binge alcohol feeding in mice. CBD or vehicle was administered daily throughout the alcohol feeding study. At the conclusion of the feeding protocol, serums samples, livers or isolated neutrophils were utilized for molecular biology, biochemistry and pathology analysis. CBD significantly attenuated the alcohol feeding-induced serum transaminase elevations, hepatic inflammation (mRNA expressions of TNFα, MCP1, IL1β, MIP2 and E-Selectin, and neutrophil accumulation), oxidative/nitrative stress (lipid peroxidation, 3-nitrotyrosine formation, and expression of reactive oxygen species generating enzyme NOX2). CBD treatment also attenuated the respiratory burst of neutrophils isolated from chronic plus binge alcohol fed mice or from human blood, and decreased the alcohol-induced increased liver triglyceride and fat droplet accumulation. Furthermore, CBD improved alcohol-induced hepatic metabolic dysregulation and steatosis by restoring changes in hepatic mRNA or protein expression of ACC-1, FASN, PPARα, MCAD, ADIPOR-1, and mCPT-1. Thus, CBD may have therapeutic potential in the treatment of alcoholic liver diseases associated with inflammation, oxidative stress and steatosis, which deserves exploration in human trials.

Hepatic overproduction of 13-HODE due to ALOX15 upregulation contributes to alcohol-induced liver injury in mice

Chronic alcohol feeding causes lipid accumulation and apoptosis in the liver. This study investigated the role of bioactive lipid metabolites in alcohol-induced liver damage and tested the potential of targeting arachidonate 15-lipoxygenase (ALOX15) in treating alcoholic liver disease (ALD). Results showed that chronic alcohol exposure induced hepatocyte apoptosis in association with increased hepatic 13-HODE. Exposure of 13-HODE to Hepa-1c1c7 cells induced oxidative stress, ER stress and apoptosis. 13-HODE also perturbed proteins related to lipid metabolism. HODE-generating ALOX15 was up-regulated by chronic alcohol exposure. Linoleic acid, but not ethanol or acetaldehyde, induced ALOX15 expression in Hepa-1c1c7 cells. ALOX15 knockout prevented alcohol-induced liver damage via attenuation of oxidative stress, ER stress, lipid metabolic disorder, and cell death signaling. ALOX15 inhibitor (PD146176) treatment also significantly alleviated alcohol-induced oxidative stress, lipid accumulation and liver damage. These results demonstrated that activation of ALOX15/13-HODE circuit critically mediates the pathogenesis of ALD. This study suggests that ALOX15 is a potential molecular target for treatment of ALD.

Phosphodiesterase 4b expression plays a major role in alcohol-induced neuro-inflammation

It is increasingly evident that alcohol-induced, gut-mediated peripheral endotoxemia plays a significant role in glial cell activation and neuro-inflammation. Using a mouse model of chronic alcohol feeding, we examined the causal role of endotoxin- and cytokine-responsive Pde4 subfamily b (Pde4b) expression in alcohol-induced neuro-inflammation. Both pharmacologic and genetic approaches were used to determine the regulatory role of Pde4b.In C57Bl/6 wild type (WT) alcohol fed (WT-AF) animals, alcohol significantly induced peripheral endotoxemia and Pde4b expression in brain tissue, accompanied by a decrease in cAMP levels. Further, along with Pde4b, there was a robust activation of astrocytes and microglia accompanied by significant increases in the inflammatory cytokines (Tnfα, Il-1β, Mcp-1 and Il-17) and the generalized inflammatory marker Cox-2. At the cellular level, alcohol and inflammatory mediators, particularly LPS, Tnfα and Hmgb1 significantly activated microglial cells (Iba-1 expression) and selectively induced Pde4b expression with a minimal to no change in Pde4a and d isoforms. In comparison, the alcohol-induced decrease in brain cAMP levels was completely inhibited in WT mice treated with the Pde4 specific pharmacologic inhibitor rolipram and in Pde4b−/− mice. Moreover, all the observed markers of alcohol-induced brain inflammation were markedly attenuated. Importantly, glial cell activation induced by systemic endotoxemia (LPS administration) was also markedly decreased in Pde4b−/− mice. Taken together, these findings strongly support the notion that Pde4b plays a critical role in coordinating alcohol-induced, peripheral endotoxemia mediated neuro-inflammation and could serve as a significant therapeutic target.

Alcohol-induced miR-155 and HDAC11 inhibit negative regulators of the TLR4 pathway and lead to increased LPS responsiveness of Kupffer cells in alcoholic liver disease.

Inflammation promotes the progression of alcoholic liver disease. Alcohol sensitizes KCs to gut-derived endotoxin (LPS); however, signaling pathways that perpetuate inflammation in alcoholic liver disease are only partially understood. We found that chronic alcohol feeding in mice induced miR-155, an inflammatory miRNA in isolated KCs. We hypothesized that miR-155 might increase the responsiveness of KCs to LPS via targeting the negative regulators of LPS signaling. Our results revealed that KCs that were isolated from alcohol-fed mice showed a decrease in IRAK-M, SHIP1, and PU.1, and an increase in TNF-α levels. This was specific to KCs, as no significant differences were observed in these genes in hepatocytes. We found a causal effect of miR-155 deficiency on LPS responsiveness, as KCs that were isolated from miR-155 KO mice showed a greater induction of IRAK-M, SHIP1, and suppressor of cytokine signaling 1 after LPS treatment. C/EBPβ, a validated miR-155 target, stimulates IL-10 transcription. We found a higher induction of C/EBPβ and IL-10 in KCs that were isolated from miR-155 KO mice after LPS treatment. Gain- and loss-of-function studies affirmed that alcohol-induced miR-155 directly regulates IRAK-M, SHIP1, suppressor of cytokine signaling 1, and C/EBPβ, as miR-155 inhibition increased and miR-155 overexpression decreased these genes in LPS or alcohol-pretreated wild-type KCs. HDAC11, a regulator of IL-10, was significantly increased and IL-10 was decreased in KCs that were isolated from alcohol-fed mice. Functionally, knockdown of HDAC11 with small interfering RNA resulted in an IL-10 increase in LPS or alcohol-pretreated Mϕ. We found that acetaldehyde and NF-κB pathways regulate HDAC11 levels. Collectively, our results indicate that the alcohol-induced responsiveness of KCs to LPS, in part, is governed by miR-155 and HDAC11.

Chronic alcohol feeding potentiates hormone-induced calcium signalling in hepatocytes.

Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined. We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca2+ -mobilizing hormones resulting in a leftward shift in the concentration-response relationship and the transition from oscillatory to more sustained and prolonged Ca2+ increases. Our data demonstrate that alcohol-dependent adaptation in the Ca2+ signalling pathway occurs at the level of hormone-induced inositol 1,4,5 trisphosphate (IP3 ) production and does not involve changes in the sensitivity of the IP3 receptor or size of internal Ca2+ stores. We suggest that prolonged and aberrant hormone-evoked Ca2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol-induced hepatocyte injury. ABSTRACT: 'Adaptive' responses of the liver to chronic alcohol consumption may underlie the development of cell and tissue injury. Alcohol administration can perturb multiple signalling pathways including phosphoinositide-dependent cytosolic calcium ([Ca2+ ]i ) increases, which can adversely affect mitochondrial Ca2+ levels, reactive oxygen species production and energy metabolism. Our data indicate that chronic alcohol feeding induces a leftward shift in the dose-response for Ca2+ -mobilizing hormones resulting in more sustained and prolonged [Ca2+ ]i increases in both cultured hepatocytes and hepatocytes within the intact perfused liver. Ca2+ increases were initiated at lower hormone concentrations, and intercellular calcium wave propagation rates were faster in alcoholics compared to controls. Acute alcohol treatment (25mm) completely inhibited hormone-induced calcium increases in control livers, but not after chronic alcohol-feeding, suggesting desensitization to the inhibitory actions of ethanol. Hormone-induced inositol 1,4,5 trisphosphate (IP3 ) accumulation and phospholipase C (PLC) activity were significantly potentiated in hepatocytes from alcohol-fed rats compared to controls. Removal of extracellular calcium, or chelation of intracellular calcium did not normalize the differences in hormone-stimulated PLC activity, indicating calcium-dependent PLCs are not upregulated by alcohol. We propose that the liver 'adapts' to chronic alcohol exposure by increasing hormone-dependent IP3 formation, leading to aberrant calcium increases, which may contribute to hepatocyte injury.

Alcohol Injury Damages Intestinal Stem Cells.

Alcohol consumption is associated with intestinal injury including intestinal leakiness and the risk of developing progressive gastrointestinal cancer. Alcoholics have disruption of intestinal barrier dysfunction that persists weeks after stopping alcohol intake, and this occurs in spite of the fact that intestinal epithelial cells turn over every 3 to 5days. The renewal and functional regulation of the intestinal epithelium largely relies on intestinal stem cells (ISCs). Chronic inflammation and tissue damage in the intestine can injure stem cells including accumulation of mutations that may result in ISC dysfunction and transformation. ISCs are a key element in intestinal function and pathology; however, very little is known about the effects of alcohol on ISCs. We hypothesize that dysregulation of ISCs is one mechanism by which alcohol induces long-lasting intestinal damage. In Vivo: Small intestinal samples from alcohol- and control-fed mice were assessed for ISC markers (Lgr5 and Bmi1) and the changes of the β-catenin signaling using immunofluorescent microscopy, Western blotting, and RT-PCR. Ex Vivo: Organoids were generated from small intestine tissue and subsequently exposed to alcohol and analyzed for ISC markers, β-catenin signaling. Chronic alcohol consumption significantly decreased the expression of stem cell markers, Bmi1 in the small intestine of the alcohol-fed mice and also resulted in dysregulation of the β-catenin signaling-an essential regulator of its target gene Lgr5 and ISC function. Exposure of small intestine-derived organoids to 0.2% alcohol significantly reduced the growth of the organoids, including budding, and total surface area of the organoid cultures. Alcohol also significantly decreased the expression of Lgr5, p-β-catenin (ser552), and Bmi1 in the organoid model. Both chronic alcohol feeding and acute exposure of alcohol resulted in ISC dysregulation which might be one mechanism for alcohol-induced long-lasting intestinal damage.

β-Caryophyllene protects against alcoholic steatohepatitis by attenuating inflammation and metabolic dysregulation in mice.

β-Caryophyllene (BCP) is a plant-derived FDA approved food additive with anti-inflammatory properties. Some of its beneficial effects in vivo are reported to involve activation of cannabinoid CB2 receptors that are predominantly expressed in immune cells. Here, we evaluated the translational potential of BCP using a well-established model of chronic and binge alcohol-induced liver injury. In this study, we investigated the effects of BCP on liver injury induced by chronic plus binge alcohol feeding in mice in vivo by using biochemical assays, real-time PCR and histology analyses. Serum and hepatic BCP levels were also determined by GC/MS. Chronic treatment with BCP alleviated the chronic and binge alcohol-induced liver injury and inflammation by attenuating the pro-inflammatory phenotypic `M1` switch of Kupffer cells and by decreasing the expression of vascular adhesion molecules intercellular adhesion molecule 1, E-Selectin and P-Selectin, as well as the neutrophil infiltration. It also beneficially influenced hepatic metabolic dysregulation (steatosis, protein hyperacetylation and PPAR-α signalling). These protective effects of BCP against alcohol-induced liver injury were attenuated in CB2 receptor knockout mice, indicating that the beneficial effects of this natural product in liver injury involve activation of these receptors. Following acute or chronic administration, BCP was detectable both in the serum and liver tissue homogenates but not in the brain. Given the safety of BCP in humans, this food additive has a high translational potential in treating or preventing hepatic injury associated with oxidative stress, inflammation and steatosis. This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Mitochondrial remodeling in the liver following chronic alcohol feeding to rats

The feeding of alcohol orally (Lieber-DeCarli diet) to rats has been shown to cause declines in mitochondrial respiration (state III), decreased expression of respiratory complexes, and decreased respiratory control ratios (RCR) in liver mitochondria. These declines and other mitochondrial alterations have led to the hypothesis that alcohol feeding causes “mitochondrial dysfunction” in the liver. If oral alcohol feeding leads to mitochondrial dysfunction, one would predict that increasing alcohol delivery by intragastric (IG) alcohol feeding to rats would cause greater declines in mitochondrial bioenergetics in the liver. In this study, we examined the mitochondrial alterations that occur in rats fed alcohol both orally and intragastrically. Oral alcohol feeding decreased glutamate/malate-, acetaldehyde- and succinate-driven state III respiration, RCR, and expression of respiratory complexes (I, III, IV, V) in liver mitochondria, in agreement with previous results. IG alcohol feeding, on the other hand, caused a slight increase in glutamate/malate-driven respiration, and significantly increased acetaldehyde-driven respiration in liver mitochondria. IG feeding also caused liver mitochondria to experience a decline in succinate-driven respiration, but these decreases were smaller than those observed with oral alcohol feeding. Surprisingly, oral and IG alcohol feeding to rats increased mitochondrial respiration using other substrates, including glycerol-3-phosphate (which delivers electrons from cytoplasmic NADH to mitochondria) and octanoate (a substrate for beta-oxidation). The enhancement of glycerol-3-phosphate- and octanoate-driven respiration suggests that liver mitochondria remodeled in response to alcohol feeding. In support of this notion, we observed that IG alcohol feeding also increased expression of mitochondrial glycerol phosphate dehydrogenase-2 (GPD2), transcription factor A (TFAM), and increased mitochondrial NAD+-NADH and NADP+-NADPH levels in the liver. Our findings suggest that mitochondrial dysfunction represents an incomplete picture of mitochondrial dynamics that occur in the liver following alcohol feeding. While alcohol feeding causes some mitochondrial dysfunction (i.e. succinate-driven respiration), our work suggests that the major consequence of alcohol feeding is mitochondrial remodeling in the liver as an adaptation. This mitochondrial remodeling may play an important role in the enhanced alcohol metabolism and other adaptations in the liver that develop with alcohol intake.

The effect of chronic alcohol consumption on mitochondrial calcium handling in hepatocytes.

The damage to liver mitochondria is universally observed in both humans and animal models after excessive alcohol consumption. Acute alcohol treatment has been shown to stimulate calcium (Ca2+) release from internal stores in hepatocytes. The resultant increase in cytosolic Ca2+ is expected to be accumulated by neighboring mitochondria, which could potentially lead to mitochondrial Ca2+ overload and injury. Our data indicate that total and free mitochondrial matrix Ca2+ levels are, indeed, elevated in hepatocytes isolated from alcohol-fed rats compared with their pair-fed control littermates. In permeabilized hepatocytes, the rates of mitochondrial Ca2+ uptake were substantially increased after chronic alcohol feeding, whereas those of mitochondrial Ca2+ efflux were decreased. The changes in mitochondrial Ca2+ handling could be explained by an up-regulation of the mitochondrial Ca2+ uniporter and loss of a cyclosporin A-sensitive Ca2+ transport pathway. In intact cells, hormone-induced increases in mitochondrial Ca2+ declined at slower rates leading to more prolonged elevations of matrix Ca2+ in the alcohol-fed group compared with controls. Moreover, treatment with submaximal concentrations of Ca2+-mobilizing hormones markedly increased the levels of mitochondrial reactive oxygen species (ROS) in hepatocytes from alcohol-fed rats, but did not affect ROS levels in controls. The changes in mitochondrial Ca2+ handling are expected to buffer and attenuate cytosolic Ca2+ increases induced by acute alcohol exposure or hormone stimulation. However, these alterations in mitochondrial Ca2+ handling may also lead to Ca2+ overload during cytosolic Ca2+ increases, which may stimulate the production of mitochondrial ROS, and thus contribute to alcohol-induced liver injury.

A mouse model of alcoholic liver fibrosis-associated acute kidney injury identifies key molecular pathways

Clinical data strongly indicate that acute kidney injury (AKI) is a critical complication in alcoholic hepatitis, an acute-on-chronic form of liver failure in patients with advanced alcoholic fibrosis. Development of targeted therapies for AKI in this setting is hampered by the lack of an animal model. To enable research into molecular drivers and novel therapies for fibrosis- and alcohol-associated AKI, we aimed to combine carbon tetrachloride (CCl4)-induced fibrosis with chronic intra-gastric alcohol feeding. Male C57BL/6J mice were administered a low dose of CCl4 (0.2ml/kg 2× week/6weeks) followed by alcohol intragastrically (up to 25g/kg/day for 3weeks) and with continued CCl4. We observed that combined treatment with CCl4 and alcohol resulted in severe liver injury, more pronounced than using each treatment alone. Importantly, severe kidney injury was evident only in the combined treatment group. This mouse model reproduced distinct pathological features consistent with AKI in human alcoholic hepatitis. Transcriptomic analysis of kidneys revealed profound effects in the combined treatment group, with enrichment for damage-associated pathways, such as apoptosis, inflammation, immune-response and hypoxia. Interestingly, Havcr1 and Lcn2, biomarkers of AKI, were markedly up-regulated. Overall, this study established a novel mouse model of fibrosis- and alcohol-associated AKI and identified key mechanistic pathways.

Decreased Whole-Body Fat Mass Produced by Chronic Alcohol Consumption is Associated with Activation of S6K1-Mediated Protein Synthesis and Increased Autophagy in Epididymal White Adipose Tissue.

Chronic alcohol consumption leads to a loss of white adipose tissue (WAT) but the underlying mechanisms for this lipodystrophy are not fully elucidated. This study tested the hypothesis that the reduction in WAT mass in chronic alcohol-fed mice is associated with a decreased protein synthesis specifically related to impaired function of mammalian target of rapamycin (mTOR). Adult male mice were provided an alcohol-containing liquid diet for 24weeks or an isonitrogenous isocaloric control diet. In vivo protein synthesis was determined at this time and thereafter epididymal WAT (eWAT) was excised for analysis of signal transduction pathways central to controling protein synthesis and degradation. While chronic alcohol feeding decreased whole-body and eWAT mass, this was associated with a discordant increase in protein synthesis in eWAT. This increase was not associated with a change in mTOR, 4E-BP1, Akt, or PRAS40 phosphorylation. Instead, a selective increase in phosphorylation of S6K1 and its downstream substrates, S6 and eIF4B was detected in alcohol-fed mice. Alcohol also increased eEF2K phosphorylation and decreased eEF2 phosphorylation consistent with increased translation elongation. Alcohol increased Atg12-5, LC3B-I and -II, and ULK1 S555 phosphorylation, suggesting increased autophagy, while markers of apoptosis (cleaved caspase-3 and -9, and PARP) were unchanged. Lipolytic enzymes (ATGL and HSL phosphorylation) were increased and lipogenic regulators (PPARγ and C/EBPα) were decreased in eWAT by alcohol. Although alcohol increased TNF-α, IL-6, and IL-1β mRNA, no change in key components of the NLRP3 inflammasome (NLRP3, ACS, and cleaved caspase-1) was detected suggesting alcohol did not increase pyroptosis. Plasma insulin did not differ between groups. These results demonstrate that the alcohol-induced decrease in whole-body fat mass resulted in part from activation of autophagy in eWAT as protein synthesis was increased and mediated by the specific increase in the activity of S6K1.

Uptake of ascorbic acid by pancreatic acinar cells is negatively impacted by chronic alcohol exposure.

Vitamin C (ascorbic acid, AA) is indispensable for normal metabolism of all mammalian cells including pancreatic acinar cells (PACs). PACs obtain AA from their surroundings via transport across the cell membrane. Chronic alcohol exposure negatively affects body AA homeostasis; it also inhibits uptake of other micronutrients into PACs, but its effect on AA uptake is not clear. We examined this issue using both in vitro (266-6 cells) and in vivo (mice) models of chronic alcohol exposure. First, we determined the relative expression of the AA transporters 1 and 2 [i.e., sodium-dependent vitamin C transporter-1 (SVCT-1) and SVCT-2] in mouse and human PACs and found SVCT-2 to be the predominant transporter. Chronic exposure of 266-6 cells to alcohol significantly inhibited AA uptake and caused a marked reduction in SVCT-2 expression at the protein, mRNA, and heterogeneous nuclear RNA (hnRNA) levels. Similarly, chronic alcohol feeding of mice significantly inhibited AA uptake and caused a marked reduction in level of expression of the SVCT-2 protein, mRNA, and hnRNA. These findings suggest possible involvement of transcriptional mechanism(s) in mediating chronic alcohol effect on AA uptake by PACs. We also observed significant epigenetic changes (histone modifications) in the Slc23a2 gene (reduction in H3K4me3 level and an increase in H3K27me3 level) in the alcohol-exposed 266-6 cells. These findings show that chronic alcohol exposure inhibits PAC AA uptake and that the effect is mediated, in part, at the level of transcription of the Slc23a2 gene and may involve epigenetic mechanism(s).

Effect of Chronic Alcohol Exposure on Physiological and Molecular Aspects of Pancreatic Acinar Cells Ascorbic Acid Transport

Vitamin C (ascorbic acid, AA) is required for normal cellular metabolism including that of pancreatic acinar cells (PAC). The vitamin is a potent antioxidant and acts as a cofactor for several important enzymes, while low intracellular level of AA leads to oxidative stress. PAC obtain the vitamin from their surroundings via transport across their cell membrane. A negative effect of chronic alcohol exposure on uptake of AA that is important for reducing cellular oxidative stress may contribute to the known deleterious effects of alcohol on the pancreas. Recent studies have shown that such alcohol exposure affects PAC uptake of other micronutrients, however its effect on PAC AA uptake is not clear. We address this issue in the present study using mouse‐derived pancreatic acinar 266‐6 cells (in vitro exposure; 50 mM ethanol for 96hrs) and mice fed alcohol chronically and controls were pair‐fed the same diet without alcohol (in vivo exposure; 25%, v/v for 4 weeks). We first determined the relative expression of the two known AA transporters in mouse and human PAC, the sodium‐dependent vitamin C transporter‐1 and ‐2 (SVCT‐1 and SVCT‐2; products of the SLC23A1 and SLC23A2 genes, respectively) and found SVCT‐2 to be the predominant transporter in PAC. Chronic exposure of 266‐6 cells to alcohol significantly inhibited AA uptake, and this inhibition was associated with a marked reduction in SVCT‐2 expression at the protein, mRNA, and heterogenous nuclear RNA (hnRNA) levels. Also, chronic alcohol feeding of mice led to a significant reduction in the level of expression of SVCT‐2 protein, mRNA and hnRNA in PAC. These findings suggest the possible involvement of transcriptional mechanism(s) in mediating the chronic alcohol effect and this observation was supported by the significant epigenetic changes (histone modifications) affecting the Slc23a2 promoter (a decrease in euchromatin H3K4me3 and an increase in heterochromatin H3K27me3) that we found in the alcohol exposed 266‐6 cells compared to controls. No significant changes in the methylation status of the predicted CpG island within the Slc23a2 promoter was found. Collectively, these findings show that chronic alcohol exposure inhibits AA uptake by PAC and that the effect is in part mediated at the level of transcription of the Slc23a2 gene and involves epigenetic mechanism(s).Support or Funding InformationSupported by grants from the DVA and the NIH (AA18071, DK56061, and DK58057).

Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury.

Chronic alcohol exposure increased hepatic receptor-interacting protein kinase (RIP) 3 expression and necroptosis in the liver but its mechanisms are unclear. In the present study, we demonstrated that chronic alcohol feeding plus binge (Gao-binge) increased RIP3 but not RIP1 protein levels in mouse livers. RIP3 knockout mice had decreased serum alanine amino transferase activity and hepatic steatosis but had no effect on hepatic neutrophil infiltration compared with wild type mice after Gao-binge alcohol treatment. The hepatic mRNA levels of RIP3 did not change between Gao-binge and control mice, suggesting that alcohol-induced hepatic RIP3 proteins are regulated at the posttranslational level. We found that Gao-binge treatment decreased the levels of proteasome subunit alpha type-2 (PSMA2) and proteasome 26S subunit, ATPase 1 (PSMC1) and impaired hepatic proteasome function. Pharmacological or genetic inhibition of proteasome resulted in the accumulation of RIP3 in mouse livers. More importantly, human alcoholics had decreased expression of PSMA2 and PSMC1 but increased protein levels of RIP3 compared with healthy human livers. Moreover, pharmacological inhibition of RIP1 decreased Gao-binge-induced hepatic inflammation, neutrophil infiltration and NF-κB subunit (p65) nuclear translocation but failed to protect against steatosis and liver injury induced by Gao-binge alcohol. In conclusion, results from this study suggest that impaired hepatic proteasome function by alcohol exposure may contribute to hepatic accumulation of RIP3 resulting in necroptosis and steatosis while RIP1 kinase activity is important for alcohol-induced inflammation.

Lactobacillus rhamnosus CCFM1107 treatment ameliorates alcohol-induced liver injury in a mouse model of chronic alcohol feeding.

Lactobacillus rhamnosus CCFM1107 was screened for high antioxidative activity from 55 lactobacilli. The present study attempted to explore the protective properties of L. rhamnosus CCFM1107 in alcoholic liver injury. A mouse model was induced by orally feeding alcohol when simultaneously treated with L. rhamnosus CCFM1107, the drug Hu-Gan- Pian (HGP), L. rhamnosus GG (LGG), and L. plantarum CCFM1112 for 3 months. Biochemical analysis was performed for both serum and liver homogenate. Detailed intestinal flora and histological analyses were also carried out. Our results indicated that the administration of L. rhamnosus CCFM1107 significantly inhibited the increase in the levels of serum aminotransferase and endotoxin, as well as the levels of triglyceride (TG) and cholesterol (CHO) in the serum and in the liver. Glutathione (GSH), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were elevated while the levels of malondialdehyde (MDA) were decreased. The enteric dysbiosis caused by alcohol was restored by increasing the numbers of both lactobacilli and bifidobacteria and decreasing the numbers of both enterococci and enterobacter. Histological analysis confirmed the protective effect of L. rhamnosus CCFM1107. Compared with the other lactobacilli and to the drug Hu-Gan-Pian, there is a high chance that L. rhamnosus CCFM1107 provides protective effects on alcoholic liver injury by reducing oxidative stress and restoring the intestinal flora.

Defect of mitochondrial respiratory chain is a mechanism of ROS overproduction in a rat model of alcoholic liver disease: role of zinc deficiency.

Morphological and functional alterations of hepatic mitochondria have been documented in patients with alcoholic liver disease (ALD). Our recent study demonstrated that zinc level was decreased in whole liver and mitochondria by chronic alcohol feeding. The present study was undertaken to determine whether zinc deficiency mediates alcohol-induced mitochondrial electron transport chain (ETC) defect and whether defective ETC function may lead to generation of reactive oxygen species (ROS). Male Wistar rats were pair fed with the Lieber-DeCarli control or ethanol diet for 5 mo. Chronic alcohol exposure increased hepatic triglyceride, free fatty acid, and 4-hydroxynonenal (4HNE) levels; meanwhile hepatic mitochondrial 4HNE level was also increased. Moreover, hepatic mitochondrial respiratory complexes I, III, IV, and V and hepatic ATP production were decreased by chronic alcohol exposure. Chronic alcohol feeding decreased peroxisome proliferator-activated receptor gamma coactivator-1-alpha (PGC1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), and mitochondrial DNA. HepG2 cells were treated with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) for 6 h. Zinc deficiency significantly decreased mitochondrial respiratory complexes I, III, and IV. In addition, PGC1α, NRF1, and TFAM levels as well as mitochondrial DNA were significantly decreased by TPEN treatment. Knockdown of mitochondrial respiratory complexes I, III, or IV by shRNA caused a decrease in mitochondrial membrane potential and an increase in ROS production. These results suggest that alcohol-induced hepatic zinc deficiency could inactivate mitochondrial biogenesis pathway and decrease mitochondrial DNA replication, which, in turn, decreases mitochondrial complex protein expression. The defect of mitochondrial respiratory complexes may worsen alcohol-induced ROS production.

Modulation of Intestinal Barrier and Bacterial Endotoxin Production Contributes to the Beneficial Effect of Nicotinic Acid on Alcohol-Induced Endotoxemia and Hepatic Inflammation in Rats.

Alcohol consumption causes nicotinic acid deficiency. The present study was undertaken to determine whether dietary nicotinic acid supplementation provides beneficial effects on alcohol-induced endotoxin signaling and the possible mechanisms at the gut-liver axis. Male Sprague-Dawley rats were pair-fed the Lieber-DeCarli liquid diets containing ethanol or isocaloric maltose dextrin for eight weeks, with or without dietary supplementation with 750 mg/liter nicotinic acid. Chronic alcohol feeding elevated the plasma endotoxin level and activated hepatic endotoxin signaling cascade, which were attenuated by nicotinic acid supplementation. Alcohol consumption remarkably decreased the mRNA levels of claudin-1, claudin-5, and ZO-1 in the distal intestine, whereas nicotinic acid significantly up-regulated these genes. The concentrations of endotoxin, ethanol, and acetaldehyde in the intestinal contents were increased by alcohol exposure, and niacin supplementation reduced the intestinal endotoxin and acetaldehyde levels. Nicotinic acid supplementation upregulated the intestinal genes involved in aldehyde detoxification via transcriptional regulation. These results demonstrate that modulation of the intestinal barrier function and bacterial endotoxin production accounts for the inhibitory effects of nicotinic acid on alcohol-induced endotoxemia and hepatic inflammation.

Ligustrazine prevents alcohol-induced liver injury by attenuating hepatic steatosis and oxidative stress

Alcoholic liver disease (ALD) is a major etiology of liver diseases, causing heavy health burdens personally and socially. Ligustrazine has been widely used in China due to its extensive pharmacological activities. However, the role of ligustrazine in ALD treatment remains unclear. Thus, this study is aimed to make up this gap and further uncover the potential mechanisms. The present work demonstrated that compared with the alcohol feeding group, ligustrazine-treated groups showed a clear decrease in aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and lactate dehydrogenase activities in serum, and a great improvement in liver histology. Additionally, ligustrazine reduced the number of foci containing CD45 positive cells and the expression of proteins associated with hepatic inflammation, apoptosis, and fibrosis. Further, ligustrazine obviously abolished alcohol-induced hepatic steatosis and hyperlipidemia. In addition, ligustrazine reversed alcohol-induced overexpression of sterol regulatory element-binding protein-1c and fatty acid synthase, and inhibition of peroxisome proliferator-activated receptor-alpha and carnitine palmitoyltransferase 1 in liver. Ligustrazine also ameliorated alcohol-induced increases in reactive oxygen species and malondialdehyde levels, and decreases in glutathione, superoxide dismutase, catalase, and glutathione reductase content in liver. Finally, chronic alcohol feeding inhibited the hepatic expression of nuclear factor erythroid 2-related factor 2 (Nrf2) at both mRNA and protein levels. Ligustrazine promoted Nrf2 expression and nuclear translocation in a dose-dependent manner. Collectively, for the first time, the present study demonstrated that ligustrazine remarkably improved chronic alcohol-induced liver injury by attenuating hepatic steatosis and oxidative stress. Further, Nrf2 activation might be requisite for ligustrazine to exert its protective effects.